Ocular tear growth factor-like protein

ABSTRACT

The present invention relates to a novel lacrimal gland protein (designated lacritin) and the nucleic acid sequences encoding that protein. Lacritin has activity as a growth factor on both human corneal epithelial cells and on the lacrimal acinar cells that produce it. Accordingly, one embodiment of the present invention is directed to the use of lacritin to treat Dry Eye and other disorders requiring the wetting of the eye.

RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 10/468,372 filed Aug. 19, 2003 which is a national stage applicationof PCT/US02/04971 filed Feb. 20, 2002 that claims priority under 35 USC,§119(e) to U.S. Provisional Application Ser. No. 60/269,900, filed Feb.20, 2001, the disclosures of which are incorporated herein in theirentirety.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant No. R01 EY09747 and R01 EY13143, awarded by National Institutes ofHealth. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention is directed to a novel ocular protein, designatedlacritin, and nucleic acid sequences encoding that protein. In oneembodiment of the invention compositions comprising lacritin are used toenhance corneal wound healing, and/or treat patients having deficienttear output.

BACKGROUND OF THE INVENTION

Health of the ocular surface is dependent on tear fluid secretions fromthe lacrimal gland. The lacrimal acinar cells comprising the lacrimalgland are polarized and highly differentiated tear secreting cells thatadhere to a complex periacinar basement membrane. The bulk of the apicalcell cytoplasm contains large secretory granules packed with tearproteins. Known tear proteins include: lysozyme, which plays a prominentbacteriocidal role on the corneal surface; lactoferrin, which functionsas both a bacteriocidal agent and as a potential inhibitor of complementactivation; secretory component, which regulates the transcellularmovement of IgA into acini lumen where it acts on the corneal surface toinhibit bacterial adhesion; and tear lipocalins (tear-specificprealbumin) and growth factors TGFα, TGFβ and EGF the functions of whichare not known. In rats, peroxidase is a tear component which has servedas a convenient marker in experimental studies. Tears not only have animportant bacteriocidal role, they also keep the cornea clean andlubricated and are important for the well-being of the cornealepithelium.

When lacrimal acinar cell tear output is collectively deficient, ‘DryEye’ (also known as keratoconjunctivitis sicca [KCS]); is the result.Dry Eye is a common ocular manifestation of Sjogren's syndrome, anautoimmune disease with unknown etiology that affects millions of peopleworldwide. Most commonly affected are post-menopausal women with varyingdegrees of severity. If untreated, Dry Eye can lead to corneal abrasion,ulceration, bacterial infection and loss of vision. Molecular mechanismsunderlying the pathogenic decline of secretory output by the mainlacrimal gland are potentially multiple. Lacrimal glands of Sjogren'ssyndrome patients contain foci of B and T lymphocytes whose pathogenicexpansion, possibly due to viral insult, can destroy lacrimal acini.However, acinar volume loss often appears insufficient relative to thetheoretical overcapacity of the main lacrimal gland. Estimates suggest apotential secretory output up to ten-fold greater than is required tomaintain a normal aqueous tear film layer. Other mechanisms thereforewarrant attention, such as aberrant secretion of one or several commoncytokines that may directly or indirectly alter lacrimal acinar cellfunction and/or lead to a decline in neural innervation. Novelautocrine/paracrine factor(s) released by lacrimal acinar cells into thetear film may be required for the health of the lacrimal secretorymachinery, ductal system and corneal epithelium. The periacinar basementmembrane is also required for normal secretory function, in part via‘BM180’ whose apparent synergy with laminin-1 promotes stimulated tearsecretion. Alteration of each of these factors, together or independentof hormonal changes, could contribute to decreased secretory capacity.

Existing protocols for treating Dry Eye suffer from several limitations.In particular, topical artificial tear replacement fluids are widelydistributed by a number of pharmaceutical companies, but the efficacy ispoor and short-lived. This lack of efficacy is due in part to the factthat constituents of natural human tears are only partially known.

The present invention is directed to a novel human extracellularglycoprotein termed ‘lacritin’ that is remarkably reduced in Sjogren'ssyndrome. Furthermore lacritin has been found to act in an autocrinemanner to enhance unstimulated (but not stimulated) tear secretion.Lacritin is produced by lacrimal acinar cells and released for the mostpart into tear fluid—much like acinar cell-expressed TGFβ's. Thisglycoprotein acts like a growth factor when added in purifiedrecombinant form to cultures of human corneal epithelial cells, and in afeedback mechanism, it also appears to act on the same lacrimal glandcells that produce it. Accordingly in one embodiment of the presentinvention, lacritin is included as an active agent in artificial tearproducts.

SUMMARY OF THE INVENTION

The present invention is directed to the isolation and characterizationof a novel lacrimal gland protein and the nucleic acid sequencesencoding that protein. Purified recombinant lacritin has activity as agrowth factor on both human corneal epithelial cells and on the lacrimalacinar cells that produce it. Accordingly, in one embodiment of thepresent invention a method is provided for treating Dry Eye and otherdisorders requiring the wetting of the eye by administering compositionscomprising a lacritin polypeptide. In addition, since the gene promoterregulating lacritin gene expression is the most specific of anypreviously described-lacrimal gland gene, the regulatory elements ofthis gene could be used to express other gene products in the eye.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation that shows recombinant lacritinenhances unstimulated secretion by isolated rat lacrimal acinar cells.Enhancement of unstimulated secretion was observed in the presence ofincreasing amounts of lacritin on lacritin-coated wells.

FIGS. 2A and 2B represent lacritin-induced proliferation and tyrosinephosphorylation. FIG. 2A is a graphic representation of the number ofhuman salivary gland (HSG) cells was determined four days afteradministering various amounts of lacritin (0 to 10 ng/ml of lacritin) toHSG cells in serum-free medium. FIG. 2B is a bar graph representing theproliferation of HSG cells upon administration of BSA (lane 1; 10 ng/ml)or serum (lane 2; 10%) was added for the same period of time. Allexperiments were performed on laminin-1-(0.05 μM) coated wells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, “nucleic acid,” “DNA,” and similar terms also includenucleic acid analogs, i.e. analogs having other than a phosphodiesterbackbone. For example, the so-called “peptide nucleic acids,” which areknown in the art and have peptide bonds instead of phosphodiester bondsin the backbone, are considered within the scope of the presentinvention.

The term “peptide” encompasses a sequence of 3 or more amino acidswherein the amino acids are naturally occurring or synthetic(non-naturally occurring) amino acids. Peptide mimetics include peptideshaving one or more of the following modifications:

-   -   1. peptides wherein one or more of the peptidyl —C(O)NR—        linkages (bonds) have been replaced by a non-peptidyl linkage        such as a —CH2-carbamate linkage (—CH2OC(O)NR—), a phosphonate        linkage, a —CH2-sulfonamide (—CH 2-S(O)2NR—) linkage, a urea        (—NHC(O)NH—) linkage, a —CH2-secondary amine linkage, or with an        alkylated peptidyl linkage (—C(O)NR—) wherein R is C1-C4 alkyl;    -   2. peptides wherein the N-terminus is derivatized to a —NRR1        group, to a —NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)2R        group, to a —NHC(O)NHR group where R and R1 are hydrogen or        C1-C4 alkyl with the proviso that R and R1 are not both        hydrogen;    -   3. peptides wherein the C terminus is derivatized to —C(O)R2        where R2 is selected from the group consisting of C1-C4 alkoxy,        and —NR3R4 where R3 and R4 are independently selected from the        group consisting of hydrogen and C1-C4 alkyl.

Naturally occurring amino acid residues in peptides are abbreviated asrecommended by the IUPAC-IUB Biochemical Nomenclature Commission asfollows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine isIle or I; Methionine is Met or M; Norleucine is Nle; Valine is Val or V;Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanineis Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine isGln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid isAsp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan isTrp or W; Arginine is Arg or R; Glycine is Gly or G, and X is any aminoacid. Other naturally occurring amino acids include, by way of example,4-hydroxyproline, 5-hydroxylysine, and the like.

Synthetic or non-naturally occurring amino acids refer to amino acidswhich do not naturally occur in vivo but which, nevertheless, can beincorporated into the peptide structures described herein. The resulting“synthetic peptide” contain amino acids other than the 20 naturallyoccurring, genetically encoded amino acids at one, two, or morepositions of the peptides. For instance, naphthylalanine can besubstituted for trytophan to facilitate synthesis. Other synthetic aminoacids that can be substituted into peptides include L-hydroxypropyl,L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl,beta.-amino acids, and isoquinolyl. D amino acids and non-naturallyoccurring synthetic amino acids can also be incorporated into thepeptides. Other derivatives include replacement of the naturallyoccurring side chains of the 20 genetically encoded-amino acids (or anyL or D amino acid) with other side chains.

As used herein, the term “conservative amino acid substitution” aredefined herein as exchanges within one of the following five groups:

-   -   I. Small aliphatic, nonpolar or slightly polar residues:        -   Ala, Ser, Thr, Pro, Gly;    -   II. Polar, negatively charged residues and their amides:        -   Asp, Asn, Glu, Gln;    -   III. Polar, positively charged residues:        -   His, Arg, Lys;    -   IV. Large, aliphatic, nonpolar residues:        -   Met Leu, Ile, Val, Cys    -   V. Large, aromatic residues:        -   Phe, Tyr, Trp

A “polylinker” is a nucleic acid sequence that comprises a series ofthree or more different restriction endonuclease recognitions sequencesclosely spaced to one another (i.e. less than 10 nucleotides betweeneach site).

As used herein, the term “vector” is used in reference to nucleic acidmolecules that has the capability of replicating autonomously in a hostcell, and optionally may be capable of transferring DNA segment(s) fromone cell to another. Vectors can be used to introduce foreign DNA intohost cells where it can be replicated (i.e., reproduced) in largequantities. Examples of vectors include plasmids, cosmids, lambda phagevectors, viral vectors (such as retroviral vectors).

As used herein a “gene” refers to the nucleic acid coding sequence aswell as the regulatory elements necessary for the DNA sequence to betranscribed into messenger RNA (MRNA) and then translated into asequence of amino acids characteristic of a specific polypeptide.

A “marker” is an atom or molecule that permits the specific detection ofa molecule comprising that marker in the presence of similar moleculeswithout such a marker. Markers include, for example radioactiveisotopes, antigenic determinants, nucleic acids available forhybridization, chromophors, fluorophors, chemiluminescent molecules,electrochemically detectable molecules, molecules that provide foraltered fluorescence-polarization or altered light-scattering andmolecules that allow for enhanced survival of an cell or organism (i.e.a selectable marker). A reporter gene is a gene that encodes for amarker.

A promoter is a DNA sequence that directs the transcription of a DNAsequence, such as the nucleic acid coding sequence of a gene. Typically,a promoter is located in the 5′ region of a gene, proximal to thetranscriptional start site of a structural gene. Promoters can beinducible (the rate of transcription changes in response to a specificagent), tissue specific (expressed only in some tissues), temporalspecific (expressed only at certain times) or constitutive (expressed inall tissues and at a constant rate of transcription).

A core promoter contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that enhance the activity or confertissue specific activity.

An “enhancer” is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.”

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the length of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially freeof contaminants normally associated with the molecule or compound in anative or natural environment.

As used herein, the term “lacritin polypeptide” and like terms refers topeptides comprising the amino acid sequence of SEQ ID NO: 4 andbiologically active fragments thereof.

As used herein, the term “biologically active fragments” or “bioactivefragment” of an lacritin polypeptide encompasses natural or syntheticportions of the amino acid sequenceMKFTTLLFLMVAGALVYAEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTAQETSMAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 4) that are capable ofspecific binding to at least one of the natural ligands of the nativelacritin polypeptide.

“Operably linked” refers to a juxtaposition wherein the components areconfigured so as to perform their usual function. Thus, controlsequences or promoters operably linked to a coding sequence are capableof effecting the expression of the coding sequence.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water and emulsions such as anoil/water or water/oil emulsion, and various types of wetting agents.

As used herein, the term “treating” includes alleviating the symptomsassociated with a specific disorder or condition and/or preventing oreliminating said symptoms.

The Invention

The present invention is directed to a novel human growth factor-likemolecule, ‘lacritin’ and compositions comprising lacritin. The inventionalso encompasses the nucleic acid sequences encoding lacritin as well asthe nucleic acid regulatory elements controlling the expression oflacritin. The full length ‘lacritin’ cDNA has been cloned from a humanlacrimal gland library (SEQ ID NO:2), and the corresponding genomic gene(SEQ ID NO: 1) has been cloned and sequenced, including 5.2 kb ofupstream and 2.8 kb of downstream genomic sequence.

In one embodiment, the present invention is directed to a purifiedpolypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ IDNO: 10, a bioactive fragment of SEQ ID NO: 4, or an amino acid sequencethat differs from SEQ ID NO: 4 by one or more conservative amino acidsubstitutions. More preferably, the purified polypeptide comprises anamino acid sequence that differs from SEQ ID NO: 4 by 20 or lessconservative amino acid substitutions, and more preferably by 10 or lessconservative amino acid substitutions. Alternatively, the polypeptidemay comprise an amino acid sequence that differs from SEQ ID NO: 4 by 1to 5 alterations, wherein the alterations are independently selectedfrom a single amino acid deletion, insertion or substitution. In onepreferred embodiment a composition is provided comprising a polypeptide,selected from the group consisting of SEQ ID NO: 4, or SEQ ID NO: 10,and a pharmaceutically acceptable carrier.

Also encompassed in the present invention are ligands that bind to thelacritin polypeptide, including the natural receptor for lacritin, aswell as methods for isolating such ligands. In one embodiment thelacritin polypeptide, or bioactive fragments thereof, is used to isolateligands that bind to the lacritin polypeptide under physiologicalconditions. The method comprises the steps of contacting the lacritinpolypeptide with a mixture of compounds under physiological conditions,removing unbound and non-specifically bound material, and isolating thecompounds that remain bound to the lacritin polypeptides. Typically, thelacritin polypeptide will be bound to a solid support using standardtechniques to allow for rapid screening of compounds. The solid supportcan be selected from any surface that has been used to immobilizebiological compounds and includes but is not limited to polystyrene,agarose, silica or nitrocellulose. In one embodiment the solid surfacecomprises functionalized silica or agarose beads. Screening for suchcompounds can be accomplished using libraries of pharmaceutical agentsand standard techniques known to the skilled practitioner.

In an alternative embodiment a cell based assay is used to detectligands that bind to lacritin (including lacritin's natural receptor).The method comprises contacting transfected cells with lacritin andisolating the relevant genes from those cells that displaylacritin-dependent calcium signaling. More particularly, in oneembodiment, previously described pools of orphan G protein coupledreceptor cDNA's will be expressed in cell lines such as HEK293T andRH7777 cells, and the transfected cells will be contacted with lacritin.A transfectant that displays lacritin-dependent calcium signaling shouldbe expressing the receptor. If the receptor is not detected in theavailable pool of orphan G protein coupled receptor cDNA's, cDNA's froma salivary ductal cell library will be transfected into 293T cells, andexpressors screened by FACS with fluorescently labeled lacritin. Inaccordance with one embodiment cells expressing receptors that can beactivated by lacritin will be detected using a cell free system. Moreparticularly, receptor activity will be detected via a GTP[_(γ35) S]binding assay using isolated cell membranes from the transfected cells.

In one aspect of the invention a method for detecting the lacritinreceptor is provided. The method comprises the steps of providing a cellthat has been transfected with nucleic acid sequences that encode forpotential cell receptors, contacting the transfected cells with lacritinand detecting those cells that display lacritin-dependent calciumsignaling. If the cells displaying lacritin-dependent calcium signalingwere transfected with more than one protein encoding gene sequence, thanthe nucleic acid sequences encoding for the lacritin receptor will beidentified by sequence analysis or other molecular technique. Forexample, the introduced recombinant nucleic acids will be isolated fromthe signaling cells and further subcloned with the resulting subclonesused to transfect cells to determine the unique sequence responsible forconferring lacritin-dependent calcium signaling to a cell.

The present invention also encompasses nucleic acid sequences thatencode the lacritin polypeptide and derivatives thereof. In particularthe present invention is directed to nucleic acid sequences comprisingthe sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or fragmentsthereof. In one embodiment, purified nucleic acids comprising at least 8contiguous nucleotides (i.e., a hybridizable portion) that are identicalto any 8 contiguous nucleotides of SEQ ID NO: 1 are provided. In otherembodiments, the nucleic acids comprises at least 25 (contiguous)nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, or 500nucleotides of SEQ ID NO: 1. In another embodiment the nucleic acidsequence comprises the sequence of SEQ ID NO: 3 or a 25 bp nucleic acidsequence that is identical to a contiguous 25 bp sequence of SEQ ID NO:3.

The present invention also includes nucleic acids that hybridize (underconditions defined herein) to all or a portion of the nucleotidesequence represented by SEQ ID NO:1 or its complement. The hybridizingportion of the hybridizing nucleic acids is typically at least 15 (e.g.,20, 25, 30, or 50) nucleotides in length. Hybridizing nucleic acids ofthe type described herein can be used, for example, as a cloning probe,a primer (e.g., a PCR primer), or a diagnostic probe. It is anticipatedthat the DNA sequence of SEQ ID NO: 1, or fragments thereof can be usedas probes to detect homologous genes from other vertebrate species.

Nucleic acid duplex or hybrid stability is expressed as the meltingtemperature or Tm, which is the temperature at which a nucleic acidduplex dissociates into its component single stranded DNAs. This meltingtemperature is used to define the required stringency conditions.Typically a 1% mismatch results in a 1° C. decrease in the Tm, and thetemperature of the final wash in the hybridization reaction is reducedaccordingly (for example, if two sequences having >95% identity, thefinal wash temperature is decreased from the Tm by 5° C.). In practice,the change in Tm can be between 0.5° C. and 1.5° C. per 1% mismatch.

The present invention is directed to the nucleic acid sequence of SEQ IDNO: 1 and nucleic acid sequences that hybridize to that sequence (orfragments thereof) under stringent or highly stringent conditions. Inone embodiment the invention is directed to a purified nucleic acidsequence that hybridizes to a 100 nucleotide fragment of SEQ ID NO: 1 orits complement under stringent conditions. In accordance with thepresent invention highly stringent conditions are defined as conductingthe hybridization and wash conditions at no lower than −5° C. Tm.Stringent conditions are defined as involve hybridizing at 68° C. in5×SSC/5× Denhardt's solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDSat 68° C. Moderately stringent conditions include hybridizing at 68° C.in 5×SSC/5× Denhardt's solution/1.0% SDS and washing in 3×SSC/0.1% SDSat 42° C. Additional guidance regarding such conditions is readilyavailable in the art, for example, by Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al. (eds.), 1995, Current Protocols in Molecular Biology,(John Wiley & Sons, N.Y.) at Unit 2.10.

In another embodiment of the present invention, nucleic acid sequencesencoding the lacritin polypeptide can be inserted into expressionvectors and used to transfect cells to express recombinant lacritin inthe target cells. In accordance with one embodiment, the nucleic acidsequence of SEQ ID NO: 3 are inserted into a eukaryotic expressionvector in a manner that operably links the gene sequences to theappropriate regulatory sequences, and lacritin is expressed in aeukaryotic host cell. Suitable eukaryotic host cells and vectors areknown to those skilled in the art. In particular, nucleic acid sequencesencoding lacritin may be added to a cell or cells in vitro or in vivousing delivery mechanisms such as liposomes, viral based vectors, ormicroinjection. Accordingly, one aspect of the present invention isdirected to transgenic cell lines that contain recombinant genes thatexpress the lacritin polypeptide of SEQ ID NO: 4.

The present invention is also directed to nucleic acid constructs forexpressing heterologous genes under the control of the lacritin genepromoter. In accordance with one embodiment a nucleic acid construct isprovided comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8operably linked to a heterologous gene. In accordance with oneembodiment the heterologous gene is a reporter gene that encodes for amarker. The marker can be any gene product that produces a detectablesignal and includes proteins capable of emitting light such as GreenFluorescent Protein (GFP) (Chalfie et al., 1994, Science 11:263:802-805) or luciferase (Gould et al., 1988, Anal. Biochem. 15: 175:5-13), as well as proteins that can catalyze a substrate (e.g., such asβ-galactosidase). The marker may also comprise intracellular or cellsurface proteins that are detectable by antibodies. Reporter moleculesadditionally, or alternatively, can be detected by virtue of a uniquenucleic acid sequence not normally contained within the cell.

As used herein, “GFP” refers to a member of a family of naturallyoccurring fluorescent proteins, whose fluorescence is primarily in thegreen region of the spectrum. The term includes mutant forms of theprotein with altered or enhanced spectral properties. Some of thesemutant forms are described in Cormack, et al., 1996, Gene 173: 33-38 andOrmo, 1996, Science 273:1392-1395, the entireties of which areincorporated herein by reference. The term also includes polypeptideanalogs, fragments or derivatives of GFP polypeptides which differ fromnaturally-occurring forms by the identity or location of one or moreamino acid residues, (e.g., by deletion, substitution or insertion) andwhich share some or all of the properties of the naturally occurringforms so long as they generate detectable signals (e.g., fluorescence).Wild type GFP absorbs maximally at 395 nm and emits at 509 nm. Highlevels of GFP expression have been obtained in cells ranging from yeastto human cells. The term also includes Blue Fluorescent Protein (BFP),the coding sequence for which is described in Anderson, et al., 1996,Proc. Natl. Acad. Sci. USA 93:16, 8508-8511, incorporated herein byreference.

Another embodiment of the present invention comprises antibodies thatare generated against the lacritin polypeptide. These antibodies can beformulated with standard carriers and optionally labeled to preparetherapeutic or diagnostic compositions. Antibodies to lacritin aregenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric(i.e “humanized” antibodies), single chain (recombinant), Fab fragments,and fragments produced by a Fab expression library. These antibodies canbe used as diagnostic agents for the diagnosis of conditions or diseasescharacterized by expression or overexpression of lacritin, or in assaysto monitor patients being treated for a conditions or diseasescharacterized by inappropriate lacritin expression. The antibodiesuseful for diagnostic purposes may be prepared in the same manner asthose described above for therapeutics. The antibodies may be used withor without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a marker. In accordance with oneembodiment an antibody is provided that specifically binds to theprotein of SEQ ID NO: 4, and more preferably the antibody is amonoclonal antibody.

The invention also encompasses antibodies, including anti-idiotypicantibodies, antagonists and agonists, as well as compounds or nucleotideconstructs that inhibit expression of the lacritin gene (transcriptionfactor inhibitors, antisense and ribozyme molecules, or gene orregulatory sequence replacement constructs), or promote expression oflacritin (e.g., expression constructs wherein the lactritin codingsequences, such as SEQ ID NO: 3 are operatively associated withexpression control elements such as promoters, promoter/enhancers,etc.).

The present invention also encompasses antigenic compositions forraising antibodies against lacritin. In one embodiment an antigeniccomposition is provided comprising the polypeptide of SEQ ID NO: 4 or anantigenic fragment thereof.

Lacritin has mitogenic activity, enhances unstimulated but notstimulated secretion, and promotes signaling in both lacrimal acinar andcorneal epithelial cells. Recombinant lacritin prepared in E. colispecifically and rapidly activates both human corneal epithelial cellsand mouse & rat lacrimal acinar cells—the latter in an autocrine mannerto enhance tear synthesis. Lacritin is active at ng/ml levels, andcontaminating bacterial LPS (endotoxin) is not detectable. Theactivities of purified recombinant lacritin indicate that it acts as agrowth factor on both human corneal epithelial cells and on the lacrimalacinar cells that produce it. Importantly, lacritin likely acts as agrowth factor only in the eye, and to a lesser extent in the salivarygland. These organ-specific beneficial effects can be used todramatically increase the efficacy of currently available topicallyartificial tear products.

Current tear supplements are not popular with patients, in part becausethe relief obtained from such products is very brief (less than 15 min).Examples of the tear substitution approach include the use of buffered,isotonic saline solutions, aqueous solutions containing water solublepolymers that render the solutions more viscous and thus less easilyshed by the eye. Tear reconstitution is also attempted by providing oneor more components of the tear film such as phospholipids and oils.Examples of these treatment approaches are disclosed in U.S. Pat. No.4,131,651 (Shah et al.), U.S. Pat. No. 4,370,325 (Packman), U.S. Pat.No. 4,409,205 (Shively), U.S. Pat. Nos. 4,744,980 and 4,883,658 (Holly),U.S. Pat. No. 4,914,088 (Glonek), U.S. Pat. No. 5,075,104 (Gressel etal.) and U.S. Pat. No. 5,294,607 (Glonek et al.) the disclosures ofwhich are incorporated herein. Existing ophthalmic formulations may alsoinclude TGF-beta, corticosteroids or androgens. All are non-specific forthe eye and have systemic effects. In contrast, lacritin is highlyrestricted to the eye and is a natural constituent of human tears andthe tear film.

An ophthalmic formulation comprising lacritin (for example, anartificial tear fluids containing lacritin) is highly desirable due tothe activity of lacritin and its localized effects. In accordance withone embodiment of the invention, compositions comprising lacritin areused to enhance corneal wound healing, and/or treat patients havingdeficient tear output. More particularly, lacritin is used in accordancewith one embodiment to treat Dry Eye syndromes, including Sjogren'ssyndrome and to enhance corneal wound healing by topical application ofcompositions comprising the lacritin polypeptide. In accordance with oneembodiment the composition comprises a pharmaceutically acceptablecarrier and a pharmaceutically effective amount of substantially purepolypeptide comprising the amino acid sequence of SEQ ID NO: 4 is usedto treat Dry Eye syndromes.

The lacritin compositions of the present invention can be formulatedusing standard ophthalmic components, and preferably the compositionsare formulated as solutions, suspensions and other dosage forms fortopical administration. Aqueous solutions are generally preferred, basedon ease of formulation, biological compatibility (especially in view ofthe malady to be treated, e.g., dry eye-type diseases and disorders), aswell as a patient's ability to easily administer such compositions bymeans of instilling one to two drops of the solutions in the affectedeyes. However, the compositions may also be suspensions, viscous orsemi-viscous gels, or other types of solid or semi-solid compositions.

The compositions of the present invention may include surfactants,preservative agents, antioxidants, tonicity agents, buffers,preservatives, co-solvents and viscosity building agents. Varioussurfactants useful in topical ophthalmic formulations may be employed inthe present compositions. These surfactants may aid in preventingchemical degradation of lacritin and also prevent the lacritin frombinding to the containers in which the compositions are packaged.Examples of surfactants include, but are not limited to: Cremophor.RTM.EL, polyoxyl 20 ceto stearyl ether, polyoxyl 40 hydrogenated castor oil,polyoxyl 23 lauryl ether and poloxamer 407 may be used in thecompositions. Antioxidants may be added to compositions of the presentinvention to protect the lacritin polypeptide from oxidation duringstorage. Examples of such antioxidants include, but are not limited to,vitamin E and analogs thereof, ascorbic acid and derivatives, andbutylated hydroxyanisole (BHA).

Existing artificial tears formulations can also be used aspharmaceutically acceptable carriers for the lacritin active agent. Thusin one embodiment, lacritin is used to improve existing artificial tearproducts for Dry Eye syndromes, as well as develop products to aidcorneal wound healing. Examples of artificial tears compositions usefulas carriers include, but are not limited to, commercial products, suchas Tears Naturale.RTM., Tears Naturale II.RTM., Tears NaturaleFree.RTM., and Bion Tears.RTM. (Alcon Laboratories, Inc., Fort Worth,Tex.). Examples of other phospholipid carrier formulations include thosedisclosed in U.S. Pat. No. 4,804,539 (Guo et al.), U.S. Pat. No.4,883,658 (Holly), U.S. Pat. No. 4,914,088 (Glonek), U.S. Pat. No.5,075,104 (Gressel et al.), U.S. Pat. No. 5,278,151 (Korb et al.), U.S.Pat. No. 5,294,607 (Glonek et al.), U.S. Pat. No. 5,371,108 (Korb etal.), U.S. Pat. No. 5,578,586 (Glonek et al.); the foregoing patents areincorporated herein by reference to the extent they disclosephospholipid compositions useful as phospholipid carriers of the presentinvention.

Other compounds may also be added to the ophthalmic compositions of thepresent invention to increase the viscosity of the carrier. Examples ofviscosity enhancing agents include, but are not limited to:polysaccharides, such as hyaluronic acid and its salts, chondroitinsulfate and its salts, dextrans, various polymers of the cellulosefamily; vinyl polymers; and acrylic acid polymers. In general, thephospholipid carrier or artificial tears carrier compositions willexhibit a viscosity of 1 to 400 centipoises (“cps”). Preferredcompositions containing artificial tears or phospholipid carriers andwill exhibit a viscosity of about 25 cps.

Topical ophthalmic products are typically packaged in multidose form.Preservatives are thus required to prevent microbial contaminationduring use. Suitable preservatives include: benzalkonium chloride,chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben,phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, orother agents known to those skilled in the art. Such preservatives aretypically employed at a level of from 0.001 to 1.0% w/v. Unit dosecompositions of the present invention will be sterile, but typicallyunpreserved. Such compositions, therefore, generally will not containpreservatives.

In humans, lacritin is produced by the lacrimal gland (large amounts),salivary gland (moderate), the basal cells of the corneal epithelium(based on immunostaining of human cornea by anti-lacritin antibodies;and ELISA detection of lacritin in human corneal epithelial cellcultures) and possibly in the thyroid, but not elsewhere. Lacritinenhances unstimulated but not stimulated secretion, has mitogenicactivity and promotes signaling in both lacrimal acinar and cornealepithelial cells. This glycoprotein has a highly restricted glandulardistribution, and this highly restricted expression pattern incombination with its functional attributes are evidence for its putativeautocrine/paracrine differentiative role in the lacrimal gland andneighboring ocular system. Since the gene promoter regulating lacritingene expression is the most specific of any previously describedlacrimal gland gene, the regulatory elements of this gene could be usedto express other gene products in the eye. In particular, the lacritingene promoter can be operably linked to a wide variety of exogenousgenes to regulate the expression of the gene products to the lacrimalgland and/or used as gene therapy to treat Dry Eye syndromes.

Alternatively, recombinant constructs comprising the lacritin promotercan be used to transform host cells in vitro as a means of screening foragonist and antagonist of lacritin function. In accordance with oneembodiment the lacritin gene promoter is linked to a heterologous geneand reintroduced into a patient to provide gene therapeutic treatment ofDry Eye syndromes. Simply stated, the promoter could be used toartificially drive the synthesis and secretion of tear proteins inpatients for which the normal gene control of these proteins may havebeen lost.

Physiological experiments using recombinant lacritin generated by E.coli suggests that it is likely a growth factor. Lacritin stimulatescalcium signaling in human corneal epithelial cells and in mouselacrimal acinar cells. It stimulates tyrosine phosphorylation in ratlacrimal acinar and human salivary ductal cells, and it enhances thequantity of tear proteins released from the. same acinar cells thatproduce it.

The full length ‘lacritin’ cDNA has been cloned from a human lacrimalgland library (SEQ ID NO:2), and the corresponding genomic gene (SEQ IDNO: 1) has been cloned and sequenced, including 5.2 kb of upstream and2.8 kb of downstream genomic sequence. A mouse homologous gene has alsobeen partially RT-PCR cloned, and this isolated mouse lacritin genesequence has 99% identity to the human sequence. Expression of lacritinis remarkably restricted. Fifty-two different tissue polyA+ or totalRNA's were screened and lacritin mRNA was detected only in lacrimal(very abundant), salivary (weak to moderate) and thyroid (weak) glands.A review of the literature suggests that this level of transcriptionalcontrol is unmatched by any other known lacrimal protein.

EXAMPLE 1 Isolation of the Lacritin Gene

cDNA and Genomic Cloning of Lacritin

Duplicate filters containing plaques (5×104 per filter) from each of tensublibraries of a human lacrimal gland cDNA library (Dickinson &Thiesse, 1995) were prehybridized at 42° C. for 4 hr in 5× Denhardt's,6.76×SSC, 10 mM sodium phosphate, 1 mM EDTA, 0.5% SDS and 182 μg/mlsalmon sperm DNA, and then hybridized overnight at 42° C. with one oftwo overlapping 23-mer oligonucleotides (‘S1’ [AGCTGGGGCACAGGCACCCGCAC;SEQ ID NO: 11] and ‘S2’ [GGGGTTCTGGGGCTGCAGCTGGG; SEQ ID NO: 12]) thathad been end-labeled with [32P]gATP 7000 Ci/mmole (ICN, Irvine Calif.)and purified. Final wash conditions were 2×SSC (45° C.), correspondingto 29.5° C. less than the S1 or S2 Tm (74.5° C. in 2×SSC for both).Plaques positive in both filters were picked and rescreened three timesin duplicate with each oligonucleotide, giving rise to forty-sevenclones. Each was subsequently reanalyzed at increasing wash stringency(−29.5, −24.5, −19.5, and −14.5° C. Tm). Inserts were excised intopBluescript and both strands sequenced via a Prizm 377 DNA Sequencer(Perkin-Elmer, Branchburg, N.J.; University of Virginia BiomolecularResearch Facility). Of identical clones, most common was a novelsequence lacking homology to BM180 (BestFit quality=16, vs randomquality of 17±2) from which the poly G-rich S1 and S2 oligonucleotideswere derived. Predicted was a 417 bp open reading frame, whose expectedprotein product was designated ‘lacritin’, in keeping with its lacrimalgland expression. Lacritin insert was subsequently used to screen ahuman P1 genomic library (carried out by Genome Systems Inc; St. LouisMo.) and three identical clones were obtained, as determined byrestriction digestion and Southern analysis. The largestlacritin-positive fragment (12.4 kb) was subcloned intact intopBluescript and both strands were compieteiy sequenced. Alignment andanalyses (Kumar et al, 2000) of cDNA and genomic sequence was primarilywith Unix-based (Gelstart, Gap) and web-based (FASTA, BestFit, Gap)Genetics Computer Group (Madison Wis.) software using default settingsand E values (FASTA) restricted to 5 or less. Genomic exon searching andidentification of splice sites was facilitated by the Baylor College ofMedicine Human Genome Sequencing Center web site. All nucleotidesequences have been submitted to the GenBank/EBI Data Bank withaccession numbers af238867 (cDNA) and ay005150 (genomic).

Northern Analysis

Human lacrimal and submandibular glands were obtained during autopsythrough the Southern division of the Cooperative Human Tissue Networkwithin 18 hours of death and most within 8 hours to minimize autolyticdegradation. The tenets of the Declaration of Helsinki were followed andinformed consent and full IRB approval were obtained. Donors werewithout known systemic bacterial or viral infections, and tissues werenormal as determined from cause of death, pathology reports and in mostcases histological examination. Tissues were snap frozen in liquidnitrogen after removal and stored at −85° C. until used for RNApreparation. Total RNA was extracted from 100-300 mg of tissue using acommercial version of the acidified guanidine thiocyanate/phenol method(RNazol B, Tel-Test, The Woodlands, Tex.). Purified RNA was dissolved indiethylpyrocarbonate-treated water, and the concentration and puritydetermined from the A260/280 absorption values. A ratio close to 2.0 wasconsidered acceptable. RNA integrity was initially determined byelectrophoresis of ethidium bromide-complexed RNA samples in a gelcontaining 0.22M formaldehyde. Samples that did not show prominent 28Sand 18S rRNA bands in a 1:1-2:1 ratio under UV light were rejected. Forblotting, RNA (5 μg/lane) was separated on a 0.8% agarose gel underdenaturing conditions (Laurie et al, 1989) and transferred tonitrocellulose. Also assayed were two purchased (cat # 7756-1 and7751-1; Clontech Labs, Palo Alto Calif.) Northern blots with multiplehuman fetal and adult poly A+RNA's and a dot blot (cat # 7770-1;Clontech Labs) containing fifty different human poly A+RNA's togetherwith control RNA's and DNA's. Blots were hybridized with [32P]-labeledlacritin insert, washed in 0.1×SSC, 0.1% SDS (Northern) or 2×SSC, 0.1%SDS (dot blot) at 55° C., and exposed to X-ray film. Dot blots were thenquantitated using NIH Image by measurement of pixel gray values ofindividual dots.

PCR Analysis and Chromosome Mapping

Alternative splicing was examined by RT-PCR using human submandibular orlacrimal total RNA and initial priming with oligo dT, or in a genespecific manner with lacritin reverse primer CGCTACAAGGGTATTTAAGGC (SEQID NO: 13) corresponding to nucleotides 523 to 503 from lacritin cDNA).Subsequent amplification with lacritin forward primerACTCACTCCTCATCCCAAAG (SEQ ID NO: 14; from exon 1; lacritin cDNAnucleotides 32 to 51) and reverse primer TTTTCAGCTTCTCATGCCC (SEQ ID NO:15; from exon 5; lacritin cDNA nucleotides 480 to 462) involveddenaturation for 2 min at 94° C., thirty cycles of amplification (94° C.for 30 sec, 52° C. for 30 sec & 72° C. for 1 min), and a final cycle for5 min at 72° C. PCR product was analyzed in agarose gels.

For FISH mapping (Genome Systems; St. Louis, Mo.), lacritin genomic DNAwas labeled with digoxigenin dUTP by nick translation and hybridized(50% formamide, 10% dextran, 2×SSC) to metaphase chromosomes fromPHA-stimulated human peripheral blood lymphocytes. Following washes,specific labeling was detected with fluoresceinated antidigoxigeninantibodies and DAPI, and examined in a Nikon Labophot microscope. Atotal of eighty metaphase cells were analyzed with sixty exhibitingspecific labeling. Confirmation was achieved by double labeling using a12q15 marker, and by comparison with human genome project draftsequence. Photographs were taken on a Nikon AFX at a final magnificationof 1,435×.

Results:

Lacrimal acinar cells are polarized exocrine secretory cells containingsome mRNA's that are remarkably under-represented in gene data banks andmay code for a rich array of differentiation factors—a presumptionunderlying the paired oligonucleotide screening of a little used humanlacrimal gland cDNA library. Among the clones identified by thisapproach was a novel cDNA sequence (SEQ ID NO: 2) represented by severalindependent clones and corresponding to a 760 bp transcript and thecorresponding amino acid sequence (SEQ ID NO: 4). The secreted geneproduct of this lacrimal gland-specific transcript was designated‘lacritin’. The lacritin nucleic acid sequence contains a 417 bp openreading frame that predicts a 14.3 kDa hydrophilic protein core with a19 amino acid signal peptide giving rise to a mature secretedcore-protein of 12.3 kDa with an isoelectic point of 5. Noteworthy is amoderately high level of glycosylation with six putative O-glycosylationsites between residues 52 and 64, and a single N-glycosylation site nearthe C-terminus, indicating that lacritin is a moderatelywell-glycosylated core protein much like the neuroglycan Cglycosaminoglycan binding domain and fibulin-2 amino globular domain towhich lacritin bears partial homology. Northern Blot analysis indicatesa high level of lacrimal gland specificity.

In FASTA searches of the primate database, partial homology is detectedwith the glycosaminoglycan binding region of human neuroglycan C (32%identity over 102 amino acids; BestFit quality=83 versus 37±5 whenlacritin sequence was randomized) and with the ‘cysteine-free’, possiblymucin-like, amino globular region of human fibulin-2 (30% identity over81 amino acids; BestFit quality=81 versus 38±5 for random). Although allthree are rich in O-glycosylation, positioning of serine and threonineis not strictly shared; and both lacritin and fibulin-2 lackglycosaminoglycan binding sites. Neuroglycan C (af059274) is a componentof brain extracellular matrix (anchored by transmembrane domain; Yasudaet al, 1998). Fibulin-2 (×89494) is widely dispersed in basementmembranes and stroma of embryonic and adult tissues (Sasaki et al,1999). Searches of non-primate databases pointed to modest homologieswith T. Cruzi mucin-like protein (af036464; BestFit quality=78 versus46±10); P. falciparum merozoite surface antigen 2 (u91656; BestFitquality=76 versus 53±6) and P. Taeda putative arabinogalactan protein(af101791; BestFit quality=74 versus 37±4).

No matching or homologous EST's were detected, in keeping withlacritin's abundance in human lacrimal gland and restricted expressionelsewhere. Northern analysis revealed a strong 760 bp lacrimal glandmessage, and weaker submandibular and thyroid gland messages of the samesize. No message was detected in human adult adrenal gland, testis,thymus, pancreas, small intestine or stomach; nor in human fetal brainlung, liver or kidney. Similarly, in a commercial dot blot of fiftydifferent human tissue poly A+ RNA's that excluded lacrimal gland,lacritin expression was found only in submandibular gland (‘salivarygland’), and to a lesser degree in thyroid. The lacritin coding sequencewas subcloned into pET-28b and pcDNA3.1/myc-His(+)C to generaterecombinant bacterial and mammalian (293-T cell) lacritin, respectively.Both forms of lacritin displayed anomalous migration in SDS PAGE.

EXAMPLE 2 Characterization of Lacritin Expression and Function

Preparation of Recombinant Lacritin and Anti-Lacritin Antisera

Full length lacritin cDNA was subcloned in frame into pET-28b (Novagen,Madison Wis.), with orientation confirmed by completely sequencingthrough the insert. Recombinant His-tagged lacritin was then generatedby IPTG-induction of BL-21 transformed cells, and purified from media onTalon (Clontech; Palo Alto Calif.) resin using standard denaturingprocedures. Required use of denaturing conditions for the binding stepis presumed to reflect His tag inaccessibility due to folding in theabsence of glycosylation. After elution, lacritin was extensivelydialyzed versus PBS, and the His tag was removed by thrombin cleavage.Protein quality was assessed by SDS PAGE and Western blotting withanti-His antibody (Santa Cruz Biotechnology; Santa Cruz Calif.).Lacritin displays anomalous mobility in SDS PAGE. Lack of contaminatingbacterial lipopolysaccharide was confirmed by the limulus amebocytelysate assay (MRL Reference Lab; Cypress Calif.). For analyticalcomparison, small amounts of mammalian lacritin were expressed in 293Tcells using pcDNA3.1/myc-His(+) (Invitrogen, Carlsbad Calif.) containinglacritin insert, and then purified under native conditions.

Anti-bacterial lacritin antiserum was subsequently prepared in rabbits(Covance Research Products, Denver Pa.), and assessed by ELISA (1/1000dilution) using recombinant bacterial lacritin (4 μg/ml) as coat andpreimmune serum (1/1000) as control. For immunohistochemistry, sectionsof zinc formalin-fixed, paraffin-embedded human tissues and a humantissue microarray were deparaffinized and rehydrated, and microwaveheated (20 min in 10 mM citrate buffer, pH 6.0) to expose antigen.Endogenous peroxidase was blocked, and then immunodetection wasperformed using the avidin-biotin-peroxidase complex method (VectastainElite kit, Vector Laboratories, Burlingame, Calif.) after incubationwith anti-lacritin antiserum or preimmune serum (1/1000) for one hour atroom temperature. Sections were counterstained with hematoxylin, placedin cupric sulfate, and then immersed in lithium carbonate.

Cell Function Analysis

Freshly isolated rat lacrimal acinar cells, and HSG (human salivarygland) ductal and HCE (human corneal epithelial) cell lines were used tostudy lacritin function. For secretion studies, rat acinar cells wereplated serum-free overnight on wells co-coated with 0.05 μM laminin 1(to ensure adhesion) and 0 to 20 μM lacritin, or alternatively withlaminin-1 (0.05 μM) and treated the next day with serum-free mediumcontaining 0 to 162 ng/ml of soluble lacritin for four hours.Unstimulated and stimulated (carbachol 10-4 M/VIP 10-8 M) secretionswere then collected, assessed (peroxidase assay) and normalized to μgcellular DNA. To study tyrosine phosphorylation, overnight serum-freecultures of both rat lacrimal acinar and HSG cells were washed andtreated with 10 ng/ml of soluble lacritin for 0.5, 2.5, 10 and 30 min.Py(20) anti-phosphotyrosine antibody immunoprecipitation of cell lysateswas then examined in Western blots of 7% SDS PAGE gels using Py(20) andECL for detection. Calcium signaling in human corneal epithelial cellswas similarly carried out in serum-free culture (Trinkaus-Randall et al,2000; Klepeis & Trinkaus-Randall, in preparation). HCE cells were grownto confluency on glass coverslips in keratinocyte media (LifeTechnologies, Rockville Md.) containing bovine pituitary extract (30μg/ml), EGF (0.1 ng/ml) and penicillin/streptomycin, and renderedquiescent 18 hrs before loading with Fluo-3AM (2 μM; Molecular Probes,Eugene Oreg.) at 37° C. for 30 min. Using an inverted Zeiss 510 LSM forvisualization, 50 sec baseline images were first recorded. While thelaser was running, lacritin was added (final concentration 4 and 40ng/ml) and the response continually monitored every 786 msec for aminimum of 200 sec.

ECM Binding Studies

Binding studies were carried out in 96 well plates coated with 10μg/well of collagen IV, laminin-1, entactin/nidogen-1, collagen I,fibronectin, vitronectin, EGF, heparin or BMS (Matter & Laurie, 1994).Wells were washed, blocked (PBS-T), incubated with 0-30 nM lacritin (inPBS-T containing 1% BSA) for 1 hr (4° C.), washed and detected withanti-lacritin antibody (1/1000) by ELISA.

Results:

Antibodies prepared against bacterial lacritin were applied to sectionsof human lacrimal and salivary glands and to tissue microarrayscontaining formalin-fixed, paraffin embedded sections of 75 differenthuman tissues and organs (see Table I). Immunoreactivity was clearlyobserved in secretory granules of acinar cells in lacrimal and major andminor salivary glands, but was not apparent in other epithelia orstroma. Presence in thyroid was equivocal (Table I). Frequency of acinarcell staining was high in lacrimal gland, whereas only scatteredsalivary acinar cells were reactive. Immunoreactivity was also apparentin secretions within lumens of lacrimal and salivary ducts. By ELISA,lacritin was detected in human tears and to a lesser extent in saliva.

TABLE I Restricted Immunolocalization of Lacritin in Human Organs^(a)adrenal medulla − adrenal cortex − appendix − bladder − bone/marrow −brain − breast − bronchus − cerebellum − colon − epididymis − esophagus− gallbladder − ganglia − heart − kidney − lacrimal gland ++++ liver −lung − lymphatics − ovary − pancreas − parathyroid − parotid gland +periph. nerve − pituitary gland − placenta − prostate − testes − minorsalivary + sem vesicle − skel muscle − skin − small intestine − spinalcord − spleen − stomach − subman gland ++ testis − thymus − thyroidgland ? uterus/vagina − ^(a)relative intensity; not all tissues shown

Lacritin function was assessed in serum-free cultures of lacrimalacinar, salivary ductal and corneal epithelial cells using secretion(acinar), proliferation (ductal), tyrosine phosphorylation (acinar,ductal) and calcium signaling (corneal epithelial) assays. Freshlyisolated rat lacrimal acinar cells were plated on increasing amounts oflacritin (with a constant small amount of laminin 1 to ensureadherence), or on laminin-1-coated wells in which lacritin was added tothe medium. Both coated and soluble lacritin enhanced unstimulatedsecretion in a dose-dependent manner (see FIG. 1), but no effect wasobserved on the stimulated secretory pathways activated by the agonistscarbachol and VIP. These results suggest an autocrine or paracrine role,possibly via receptors on the luminal acinar cell surface. As lacritinflows from acini, it contacts ductal epithelial cells and finally thecorneal epithelium.

Quiescent human submandibular ductal (‘HSG’) cells were cultured inserum-free media containing increasing amounts of lacritin and cellproliferation was studied. The lacritin cultures looked healthier; afterfour days, a dose-dependent increase in ductal cell number was apparent(see FIG. 2 a) that reached a level more than twofold that of the BSA(10 ng/ml) negative control (see FIG. 2 b). The same level of lacritinpromoted the transient tyrosine phosphorylation of a 48 kDa band in bothHSG and rat lacrimal cells.

Next, calcium signals in human corneal epithelial cells were examined.Whereas the basal level of signaling was negligible, the addition oflacritin resulted in rapid and sustained calcium waves that propagatedthroughout the cells. Wave onset preceded that of the usual response toepidermal growth factor (20-40 sec), and the amplitude of the responsedepended on the concentration of lacritin. To ensure that bacteriallipopolysaccharide (a possible contaminant of recombinant protein preps)was not involved, samples were tested in the limulus amebocyte lysateassay; and no lipopolysaccharide was detected (<0.05 EU/ml). Finally,the ability of lacritin to bind the tear film components fibronectin orvitronectin was examined; as well as constituents of the periacinarbasement membrane that might harbor small amounts of lacritin notdetectable by the immunohistochemical procedure. Lacritin displayed aremarkable avidity for fibronectin and vitronectin, and there was astrong basement membrane binding attributable to collagen IV,nidogen/entactin and laminin-1-similar to that observed for fibulin-2(Sasaki et al, 1995). No binding was observed to collagen I, EGF orheparin.

The rather broad lacritin lacrimal gland message was suggestive ofalternatively spliced forms, or RNA degradation. The same was not truefor submandibular gland in which a discrete, but much less intensesignal was apparent. To address this issue and to gain information onhow the lacritin gene is arranged, a 12.4 kb genomic fragment wassequenced, the largest lacritin-positive fragment readily obtainablefrom the lacritin genomic clones. The gene consists of five exonspreceded by a predicted promoter sequence 109 to 59 bp upstream of thetranslation start site (promoter score=1.0; NNPP/Eukaryotic). Exon 1encodes the complete signal peptide and includes 38 bp of 5′untranslated sequence. Exon 3 contains sequence for all putative0-glycosylation sites. The predicted N-glycosylation site is formed atthe exon 4/exon 5 splice junction. Exon 5 includes 53 bp of 3′untranslated sequence. Three potential polyadenylation sites aredetected 367, 474 and 534 bp downstream of exon 5, the first of whichwould be in keeping with a 760 bp transcript. Sequences at exon-intronboundaries all conform to predicted splice donors or acceptors, with theexception of the exon 4 splice acceptor. Intronic sequences revealedcommon intronic repeat elements. Also independently discovered on aseparate genomic fragment was a lacritin pseudogene lacking 38 bp of 5′exon 1 sequence.

To examine possible alternative splicing, RT-PCR was used withsubmandibular or lacrimal gland cDNA as template and forward and reverseprimers from exons 1 and 5, respectively, each including untranslatedflanking sequence. A single PCR product was detected in both organswhose size (449 bp) was in keeping with transcription from all fiveexons without alternative splicing. FISH revealed that the lacritin geneis located on chromosome 12, a result confirmed by double labeling witha probe for 12q15. Measurement of ten specifically labeled chromosomeslocated the lacritin gene approximately 16% of the distance from thecentromere to the telomere of 12q, an area that corresponds to 12q13.Also found on 12q13 is a rare genetic alacrimia known as Triple ASyndrome. Attempted PCR using iacritin genomic primers and BAC templatesspanning the triple A syndrome region failed to produce PCR product. Thelacritin gene is partially included in draft sequences AC068789.4,AC025686.2 and AC025570.6 pointing to a 12q13 location approximately65.1 to 65.9 Mbp from the centromere.

Discovery of lacritin developed from the hypothesis that multipleextracellular factors trigger glandular differentiation, particularlygrowth factors and components of the surrounding extracellular matrix.Indeed, partial or failed acinar formation has been reported in micelacking the TGFb superfamily members or receptors, ErbB4, theprogesterone receptor, the extracellular matrix glycoproteinosteopontin, EGF receptor (with TGFa and amphiregulin), fibroblastgrowth factor receptor 2 (IIIb), or the growth factor FGF-10. Linkingsuch factors to the secretory function of acinar cells in culture hasproven more complex. Nonetheless, it is clear that the periacinarmesenchymal and hormonal environment affect glandular development andfunction, and that both autocrine and paracrine regulation playimportant roles. Most delicate are primary cultures of freshly isolatedexocrine cells, particularly lacrimal acinar cells that functionallydedifferentiate in the absence of lacrimal-1 and lower molecular massfactors derived from the extracellular matrix and elsewhere.

Introduction of recombinant lacritin to cultures of lacrimal acinar,salivary ductal and corneal epithelial cells provided interestingfunctional insights. Lacrimal acinar cells displayed enhancedunstimulated (but not stimulated) secretion and rapid tyrosinephosphorylation of a 48 kDa protein. Ductal cells phosphorylated thesame 48 kDa band and were proliferative. A rapid and sustained calciumtransient was noted in corneal epithelial cells. Thus all cell typescontributing to or benefiting from lacritin outflow appear to belacritin-inducible, whereas controls were negative and there was noevidence of contaminating bacterial lipopolysaccharide (known to beproliferative in immune cell cultures). How lacritin acts remains to beelucidated. Possibly a common receptor(s) is mediatory, ligation ofwhich may be jointly linked to tyrosine phosphorylation and calciumrelease as in neural retina where tyrosine kinases have been associatedwith capacitative calcium entry and inositol-3-phosphate induced releaseof intracellular calcium stores. Alternatively, lacritin signaling inthe three cell types may differ. Lacrimal acinar, ductal and cornealepithelial cells perform strikingly different functions. Although someintracellular signaling machinery may be common, others are unique, andsome common machinery may be put to different use. Calcium signaling inlacrimal acinar cells is most frequently a downstream effect ofmuscarinic receptor ligation that mediates the release of tear proteinsby the stimulated secretory pathway, a pathway apparently unaffected bylacritin. Yet, subtleties in calcium amplitude, frequency andlocalization, dependent on the nature and dose of the agonist, can havedramatically different effects. Contrasting lacritin is BM180, aperiacinar basement membrane constituent that appears to act only on thestimulated secretory pathway. Balancing the amounts of availablelacritin and BM180 may offer a simple mechanism by which secretorycapacity in adult and developing glands may be controlled.

Immunolocalization of lacritin in secretory granules, in secretorycontent of ducts and in tears was extended by binding studies revealinga remarkable affinity for tear constituents fibronectin and vitronectin.Though not immunodetected elsewhere, lacritin also bound the commonperiacinar basement membrane components nidogen/entactin, collagen IV,and laminin-1; but not collagen I, EGF or heparin. Similar bindingproperties have been reported for fibulin-2 (Sasaki et al, 1995).Although the significance and precise nature of these interactionsremains to be determined, basement membrane binding is perhaps analogousto growth factors whose extracellular matrix accumulation, althoughfunctionally potent, is often too low for reliable immunodetection.Alternatively, basement membrane binding (if any) could possibly occursecondary to tissue damage.

EXAMPLE 3 Characterization of the Lacritin Promoter

The working hypothesis is that lacritin gene activity is attributable toan atypically restrictive and powerful promoter working hand in handwith unique enhancer (and possibly repressor) elements in a milieu ofappropriate transcription factors and co-regulators. Suchtissue-specific transcriptional control equals or exceeds that of theaA-crystallin (lens), rhodopsin (retina), aldehyde dehydrogenase class 3and keratocan genes (cornea), and offers a unique opportunity toinitiate a new body of literature on nuclear management of geneexpression in the human lacrimal gland.

Mapping of Lacritin Gene Regulatory Elements

Elucidating how lacritin gene expression is targeted to the lacrimalgland will be determined as described below to better understandinglacrimal gene regulation. First of all the identify the lacritintranscription initiation site(s) will be confirmed experimentally. Basedon computational promoter analysis, transcription is anticipated to beinitiated at a single site located 69 bp upstream (‘−69 bp’; ‘NeuralNetwork’) of the ATG translation start site. The ‘TATA-box’ and/or‘Initiator’ (‘Inr’) elements of the core promoter play an important rolein establishing the start site of transcription in many genes,particularly those highly expressed. As an example, Inr elements at +1,+220 (also TATA-box at +190 bp) and +316 bp (intronic) designatetranscription start sites in the human keratocan gene as experimentallyconfirmed by primer extension (see Tasheva E S, Conrad A H, Conrad G W.Identification and characterization of conserved cis-regulatory elementsin the human keratocan gene promoter. Biochim Biophys Acta. 2000 Jul24;1492(2-3):452-9); and a TATA-box figures prominently in transcriptioninitiation of aA crystallin, rhodopsin, and aldehyde dehydrogenase genepromoters. If the Neural Network-predicted −94 to −46 bp region doesindeed comprise the lacritin core promoter with transcription start siteat −69 bp (score=1.0), then putative TATA-box and Inr elements at −52and −67 bp, respectively should play a key role in transcriptioninitiation. Alternatively, transcription could begin at −62 bp, assuggested by ‘CorePromoter’. Primer extension and RNA ligase-mediated5′-RACE will resolve this question.

For primer extension, advantage will be taken of a 20-mer reverse primer(‘LacP83’) designed by ‘Prime’ (GCG, Madison Wis.) which iscomplementary to nucleotides 64 to 83 bp of lacritin mRNA. As perroutine procedure, LacP83 will be end-labeled by phosphorylation with T4polynucleotide kinase in the presence of [g³²P]ATP, annealed with totallacrimal RNA (100 fmol primer per 10 μg RNA) for 20 min at 58° C.,cooled, and then incubated for 30 min at 41° C. with AMV reversetranscriptase (Promega, Madison Wis.) in the presence ofdeoxynucleotides. Size of newly formed cDNA(s), as analyzed bydenaturing SDS PAGE analysis/radiography, provides sufficientinformation to calculate the approximate transcription start sitelocation(s)—with identification of the 5′ terminus(i) determined bysemiautomatic ABI sequencing of cDNA from a scaled up non-radioactiveextension reaction and RNA ligase-mediated 5′-RACE. Primer extensioncontrols will include replacement of lacrimal RNA with total yeast RNA(or no RNA), and use of an RNA prepared by in vitro transcription withaccompanying primer (Promega, Madison Wis.) for which primer extensionconditions have been previously established.

For confirmation, RNA ligase-mediated 5′-RACE (‘GeneRacer’; Invitrogen)will be utilized. This is a powerful PCR-based modification of primerextension. For this purpose, 1-5 μg of total human lacrimal RNA will betreated with calf intestinal phosphatase (1 U per 10 μl reaction mix) toremove 5′ phosphates from degraded RNA and non-mRNA contaminants.Incubation with tobacco acid pyrophosphatase (0.5-1 U per 10 μl reactionmix) eliminates the 5′-CAP structure present only on authentic 5′-ends,and makes possible ligation of a kit-specific RNA oligonucleotide(‘GeneRacer RNA Oligo’) with T4 RNA ligase (5 U per 10 μl reaction mix).Subsequent LacP83-primed reverse transcription will generate a singlestrand cDNA. The cDNA will then be PCR amplified using LacP83 and aprimer complementary to the 5′ RNA oligo as primer pair, and sequencedto identify the start site(s). In negative PCR controls, amplificationswill be attempted in the absence of LacP83 or GeneRacer RNA Oligo orwithout template, and if banding or smearing is observed further PCRoptimization will be carried out (ie. use of less template or fewer PCRcycles or do nested PCR to increase amplicon amount, or use touchdownPCR).

It is anticipated that a single primer extended cDNA band of 152 (or145) bp will be observed in keeping with a transcription start site at−69 bp (or −62 bp) and inclusion of 83 bp from the 5′ end of the primerto the translation start site [69+83 bp (or 62+83 bp)]. This expectationis in agreement with the single transcript apparent by Northern analysisof human salivary gland. The broader human lacrimal band has beeninterpreted as attributable to MRNA abundance combined with possiblysome slight degradation. Although no alternative splicing has beenobserved, the possibility of a second transcript cannot be completelyruled out.

A luciferase reporter constructs will also be generated andtransfection-based regional mapping of lacritin gene regulatory elementswill be initiated. It is hypothesized that Bayesian alignment of humanand mouse lacritin genes will provide an excellent foundation forinterpretation of reporter construct activity, and that evolutionaryconservation similarly will make feasible utilization of a rabbitlacrimal acinar cell line as transfection host—the only immortalizedcell line from lacrimal gland of any species. This exploratory approachwill lay the conceptual groundwork for more detailed studies both invitro and in vivo.

Lacritin's tissue specificity is presumably founded in the nature andassortment of transcription factor binding modules that comprise itsgene promoter and putative enhancer region(s). Lens-preferred expressionof the aA-crystallin gene, for example, is governed by a transcriptioncomplex of CREB/CREM, aA-CRYBP1, Pax 6, TBP, USF, AP-1 (context of AP-1important for tissue specificity) and L-maf that nucleates on the 150 bpaA-crystallin promoter. Transfected plasmid constructs that artificiallyposition luciferase or chloramphenicol acetyltransferase expressionunder the control of intact or progressively 5′ shortened (or mutated)promoter regions, has been used previously to identify cis-actingregulatory region of a promoter. The versatile and sensitive‘Dual-Luciferase Reporter Assay System’ (Promega) for examplesequentially assays both the transfected gene promoter underinvestigation (as manifested by the level of expressed fireflyluciferase) and a co-transfected internal positive HSV-TK controlpromoter designed to independently drive expression of a synthetic seapansy luciferase with distinct substrate properties at a constantbaseline level (see below). Subsequent investigation in transgenic miceusing b-galactosidase as reporter brings chromosomal context into play.Recent availability of a rabbit lacrimal cell line (Nguyen D H, BeuermanR W, Halbert C L, M a Q, Sun G. Characterization of immortalized rabbitlacrimal gland epithelial cells. In Vitro Cell Dev Biol Anim. 1999April; 35(4):198-204.) and genomic cloning of lacritin now open up thisline of investigation to the lacrimal gland field.

If transcription is indeed initiated at −69 or −62 bp, upstream genomicconstructs spanning −2435 to −10 bp (‘Lacrgen2.4’), −1619 to −10 bp(‘Lacrgen1.6’) or −856 to −10 bp (‘Lacrgen0.9’) could include all ormost elements necessary for tissue specific and elevated expression.Preparation of each will take advantage of parent amplicon ‘Lacrgenlnit’(−2960 to −10 bp) to be generated by PCR from the 12.4 kb lacritingenomic fragment using reverse primer ‘LacP-10/Xho I’ (−10 to −31 bp)with an Xho I site incorporated, and forward primer ‘LacP-2960’ (−2960to −2942 bp). Primer pairs are designed by ‘Prime’ (GCG, Madison Wis.).Subsequent digestion of Lacrgenlnit with XhoI, Bgl II/Xho I or HindIII/Xho I yields fragments Lacrgen2.4, Lacrgen1.6 or Lacrgen0.9,respectively with ends suitable for ready ligation (after gelpurification) into the multiple cloning region of pGL3-Basic justupstream of the promoterless and enhancerless luciferase gene (luc+).

A new rabbit lacrimal acinar cell line (Nguyen et al, '99), that hasbeen cultured for twelve months without difficulty will be used for thetransfection studies. The cells display a strong epithelial morphologyand synthesize secretory component, transferrin and transferrinreceptor. Importantly, they also express lacritin and are readilytransfectable. To carry out transfections, ≈80% confluentserum-containing cultures in 96 well plates will be transientlytransfected with Lacrgen2.4, Lacrgen1.6 or Lacrgen0.9 in pGL3-Basic plusinternal control phRL-TK plasmid (total of 0.24 μg plasmid/well; 50:1ratio of pGL3-Basic to phRL-TK) using ≈0.8 μl/well LipofectAMINE 2000reagent (Invitrogen Life Technologies). 48 hours later, cultures will begently washed three times in PBS, lysed for 15 min in 1X ‘Passive LysisBuffer’ (20 μl/well; Promega), and assayed for firefly luciferase uponaddition of ‘Luciferase Assay Reagent II’ (100 μl/well) in an L-Max 96well plate luminometer (Molecular Devices, Menlo Calif.; online withcomputer). Readings are zeroed to similarly treated wells containinglysate of cells not transfected. Subsequently, ‘Stop & Glo Reagent’ (100μl/well) is added for assay of sea pansy (Renilla) luciferase. Inclusionof identically transfected human 293 cells will serve as a negativecontrol, whereas Araki-Sasaki human corneal epithelial cells (HCE-T) andHSG human salivary cells (both secrete lacritin) are suitable positivecontrols. Optimal lacrimal LipofectAMINE transfection, and ‘Bright-Glo’luciferase assay conditions (Promega), will take advantage of thepGL3-Control vector whereby transfected cells benefit from luc+expression under SV40 promoter and enhancer control. It is expected thattransfection efficiency will be 75-90%, and that one of Lacrgen2.4,Lacrgen1.6 or Lacrgen0.9—likely Lacrgen1.6 or Lacrgen0.9—will bestdefine the minimal sequence required for iacritin promoter activity.

This course of investigation offers a logical starting point for thegeneration and testing of Lacrgen2.4, Lacrgen1.6 or Lacrgen0.9-derivedconstructs progressively shortened 5′ by nested deletion, an approachapplied to the genomic sequencing of the lacritin gene and flankingregions. Making this possible are single Kpn I and Sac I sites justupstream of each insert in the pGL3-Basic multiple cloning region, andlack of any internal Kpn I or Sac I sites in Lacrgen2.4, Lacrgen1.6 orLacrgen0.9—the latter as determined by Map (GCG). Thus when digestedwith Kpn I and Sac I, a linear plasmid will be generated in which theKpn I end is exonuclease III resistant (3′ protruding) and the Sac I end(3′ recessed) is sensitive. Proximity of Lacrgen2.4, Lacrgen1.6 orLacrgen0.9 to the Sac I site makes them sensitive to exonucleaseshortening. To carry this out, 2 μg of plasmid construct is Kpn I andSac I digested. After enzyme inactivation (10 min at 70° C.) and coolingon ice, linear plasmid in exonuclease III buffer is treated withexonuclease III in a final volume of 40 μl at 25° C. or 15° C. such asto achieve successive 50-100 bp deletions at 3 min intervals. 2 μlaliquots of each 3 min time point are removed to tubes on ice containingS1 nuclease. After all timed aliquots have been taken, plasmid digestsare removed from ice, incubated at room temperature for 30 min for S1nuclease digestion of overhangs, heat inactivated, recircularized byblunt end ligation in the presence of T4 DNA ligase, examined in agarosegels and transformed into competent cells with ampicillin selection.Plasmid preps of each are then applied to the transfection (withinternal control plasmid) of lacrimal acinar cells, and assessment ofluciferase expression.

1. A method for identifying a nucleic acid sequence encoding a lacritinreceptor, said method comprising providing cells that have beentransfected with nucleic acid sequences that encode for potential cellreceptors; contacting said transfected cells with lacritin (SEQ IDNO:4); detecting cells that display lacritin-dependent calciumsignaling; isolating nucleic acid sequences from the detected cells; andidentifying a nucleic acid sequence that confers lacritin-dependentcalcium signaling on cells transfected with that nucleic acid sequence.